Most animals exhibit elevated levels of circulating corticosteroids in response to stress, and long term exposure to exogenous glucocorticoids leads to depressed testosterone production and sexual dysfunction. Recent evidence has shown that glucocorticoids act directly on Leydig cells by inhibiting processes critical for testosterone biosynthesis. Another body of evidence has underscored the importance of interactions between the steroid receptors and nuclear protooncogenes. Thus, we are interested in exploring the relationship between the expression of the AP-I nuclear proteins. Fos and Jun, in Leydig cells following glucocorticoid treatment. During the period covered by this proposal we will investigate the glucocorticoid- induced expression of the AP-I protooncogenes in Leydig cells utilizing the following approaches: 1) the amount and transcript size of the c-jun, jun B, and c-fos mRNAs in isolated ray Leydig cells will be determined by means of Northern blot analysis, 2) the levels of the protein products translated from these protooncogene transcripts will be determined using a Western immunoblotting analysis, 3) mobility shift assays will be used to assess potential AP-I and/or Ap-I/glucocorticoid receptor interactions and binding activities in purified Leydig cells, and 4) in vivo experiments designed to study the effect of glucocorticoid depletion on processes described in aims 1-3 using adrenalectomized animals (ADX). Data generated from this study will be used to design experiments intended to reveal more specific mechanisms underlying the glucocorticoid/AP-I regulatory pathways. For instance, promoters for genes essential to steroidogenesis will be fused to CAT vectors and, following Leydig cell transfection, utilized for transcriptional analysis. It is important that hormones released during the stress response be examined in terms of their effects on testosterone biosynthesis and potential effects on male fertility.